Cognate interactions between antigen presenting cells (APCs) and CD4+ T-cells are mediated by T-cell receptor recognition of antigenic peptide-class II complexes present on the surface of APCs. B-cell are distinct from other class II-restricted APCs such as dendritic cells and macrophages because they express a clonally-restricted antigen-specific receptor (i.e., the B-cell receptor (BCR)) via which they can process and present cognate antigen. However, B-cells can also process and present non-cognate antigen by fluid-phase (F-P) endocytosis provided that it is present as sufficiently high concentration. Using a BCR transgenic mouse model, we have analyzed these two pathways of antigen processing in normal splenic B-cells. Interestingly, unlike antigen internalized via F-P endocytosis, native BCR-internalized antigen can persist in a non-lysosomal endocytic compartment within the B-cell for over 24 hours. Moreover, this persisting BCR-internalized antigen can be processed to peptides, supporting the prolonged cell surface persistence of peptide-class II complexes on these B-cell. The goal of the first specific aim of this grant application is to better understand the cell biology behind the intracellular persistence of BCR-internalized antigen. Furthermore, using a peptide-class II complex-specific monoclonal antibody (mAb) designated C4H3, we have determined that peptide-class II complexes formed via BCR-mediated antigen processing (termed Type II peptide-class II complexes are biologically distinct from peptide-class II complexes formed via F-P antigen processing (termed Type I peptide-class Ii complexes). Specifically, C4H3 ligation of Type II vs. Type I peptide-class II complexes leads to distinct cell signaling events within the B-cell. Moreover, Type II but not Type I peptide-class II complexes are able to mediate the internalization of bound C4H3 mAb. The goal of the second specific aim of this grant application is to determine the cell biological basis for the differences in behavior between Type I vs. Type II peptide-class II complexes. The goal of the third specific aim of this grant application is to determine the immunological consequences of the differences between Type I and Type II peptide-class II complexes.